In vitro models comprising various cellular elements of brain have been established in order to identify the mechanisms involved in the observed in vivo tolerance to ischemia of the brain pretreated either by tumor necrosis factor-alpha (TNF-alpha) or by hypoxia/ ischemia. TNF-alpha pretreatment of rat cortical astrocytes and brain microvascular endothelial cells (BMEC) renders these cells unresponsive to TNF activation 24 hours later in terms of up-regulation and expression of ICAM-1 (analyzed by FACS fluorescent cell ELISA as well as by Northern blot analysis of ICAM-1 mRNA). The role of ceramide, a lipid messenger implicated in TNF signaling, has been examined in this model. The ceramide analogue, C-2 ceramide, when added to the cells for 4 hours to substitute for TNF pretreatment, failed to induce tolerance to subsequent TNF activation in both cortical astrocytes and BMEC. However, addition of C-2 ceramide 30 min before or even 1 hour after TNF treatment resulted in inhibition of up-regulation of ICAM-1 at both protein and mRNA levels. HPLC determination of endogenous ceramide concentrations in TNF-activated cortical astrocytes and BMEC demonstrated two peaks of ceramide release: an early 1.9-fold increase at 15-20 minutes, and a late 2.5-fold increase at 18-21 hours after TNF addition. Inhibition of ceramide synthase with Fumonisin B1 attenuated TNF-induced preconditioning of astrocytes in a dose-dependent manner. These results strongly suggest that late ceramide release is a mediator of TNF-induced tolerance. Primary cultures of neonatal cortical neurons were subjected to 60 minutes of hypoxia and reoxygenated for various times. Cell death was quantitated using the ethidium iodide exclusion fluorescence method. The number of dead cells gradually increased over 8 hours of reoxygenation reaching 21.9% at 8 hours of hypoxia. Pretreatment of neuronal cultures with short hypoxia (15 minutes) 24 hours prior to 60 minutes of hypoxia, protected neurons against hypoxia (number of dead cells was 9.4% versus 35% in non-pretreated cultures). Pretreatment with TNF (50 ng/ml) or ceramide (10 micromolar) also protected cortical neurons against 60 minutes of hypoxia. TNF-preconditioning can induce tolerance to subsequent TNF and hypoxia exposure in BMEC, astrocytes and cortical neurons. Ceramide appears to be involved in the intracellular signaling pathways leading to tolerance of brain cells to ischemia.